ABSTRACT Krüppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress, such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death by mouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental role for KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF2-/- embryos die earlier, by E11.5. E9.5 FVB/N KLF2-/- hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normal single layer. By E10.5, traditional and endothelial-specific FVB/N KLF2-/- AV cushions are hypocellular, suggesting that the cells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF2-/- hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number of mesenchymal cells migrating from FVB/N KLF2-/- AV explants into a collagen matrix is reduced considerably compared to wild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/N KLF2-/- embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF2-/- hearts have largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF2-/- embryos have a delay in the formation of the atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucose dehydrogenase (Ugdh), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and Ugdh promoters in chromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. In conclusion, KLF2-/- heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of important cardiovascular genes.

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Kru ¨ppel-Like Factor 2 Is Required for Normal MouseCardiac DevelopmentAditi R. Chiplunkar1, Tina K. Lung1, Yousef Alhashem1, Benjamin A. Koppenhaver1, Fadi N. Salloum2,Rakesh C. Kukreja2, Jack L. Haar3, Joyce A. Lloyd1,4*1Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, Virginia, United States of America, 2Department of Internal Medicine,Virginia Commonwealth University, Richmond, Virginia, United States of America, 3Department of Anatomy and Neurobiology, Virginia Commonwealth University,Richmond, Virginia, United States of America, 4Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, United States of AmericaAbstractKru ¨ppel-like factor 2 (KLF2) is expressed in endothelial cells in the developing heart, particularly in areas of high shear stress,such as the atrioventricular (AV) canal. KLF2 ablation leads to myocardial thinning, high output cardiac failure and death bymouse embryonic day 14.5 (E14.5) in a mixed genetic background. This work identifies an earlier and more fundamental rolefor KLF2 in mouse cardiac development in FVB/N mice. FVB/N KLF22/2 embryos die earlier, by E11.5. E9.5 FVB/N KLF22/2hearts have multiple, disorganized cell layers lining the AV cushions, the primordia of the AV valves, rather than the normalsingle layer. By E10.5, traditional and endothelial-specific FVB/N KLF22/2 AV cushions are hypocellular, suggesting that thecells accumulating at the AV canal have a defect in endothelial to mesenchymal transformation (EMT). E10.5 FVB/N KLF22/2 hearts have reduced glycosaminoglycans in the cardiac jelly, correlating with the reduced EMT. However, the number ofmesenchymal cells migrating from FVB/N KLF22/2 AV explants into a collagen matrix is reduced considerably compared towild-type, suggesting that the EMT defect is not due solely to abnormal cardiac jelly. Echocardiography of E10.5 FVB/NKLF22/2 embryos indicates that they have abnormal heart function compared to wild-type. E10.5 C57BL/6 KLF22/2 heartshave largely normal AV cushions. However, E10.5 FVB/N and C57BL/6 KLF22/2 embryos have a delay in the formation ofthe atrial septum that is not observed in a defined mixed background. KLF2 ablation results in reduced Sox9, UDP-glucosedehydrogenase (Ugdh), Gata4 and Tbx5 mRNA in FVB/N AV canals. KLF2 binds to the Gata4, Tbx5 and Ugdh promoters inchromatin immunoprecipitation assays, indicating that KLF2 could directly regulate these genes. In conclusion, KLF22/2heart phenotypes are genetic background-dependent. KLF2 plays a role in EMT through its regulation of importantcardiovascular genes.Citation: Chiplunkar AR, Lung TK, Alhashem Y, Koppenhaver BA, Salloum FN, et al. (2013) Kru ¨ppel-Like Factor 2 Is Required for Normal Mouse CardiacDevelopment. PLoS ONE 8(2): e54891. doi:10.1371/journal.pone.0054891Editor: Elena Aikawa, Brigham and Women’s Hospital, Harvard Medical School, United States of AmericaReceived July 5, 2012; Accepted December 18, 2012; Published February 14, 2013Copyright: ? 2013 Chiplunkar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This work was supported by the National Institutes of Health (NIH R01 DK074694). The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: jlloyd@vcu.eduIntroductionCongenital heart defects (CHDs) are a leading cause of infantmorbidity and mortality (reviewed in [1]). Valve and septal defectsaccount for the majority of CHDs. Mutations in transcriptionfactor genes, including Nkx2-5 [2] and the cardiac T-box geneTBX5 [3], are important for normal valve development inhumans. Mutations or variants in other transcription factor genesare likely to be involved in valve defects.During mouse heart development, localized swellings of theendocardial layer arise at approximately embryonic day 9.5 (E9.5),and form the endocardial cushions of the atrioventricular (AV)canal and the outflow tract. The endocardial cushions are formedby endothelial to mesenchymal transformation (EMT). DuringEMT, AV endocardial cushion cells undergo hypertrophy, loss ofcell-cell contacts, lateral mobility, formation of mesenchymal-likecell processes (filopodia), and migration into the cardiac jelly(Reviewed in [4]). Without normal cardiac jelly, endothelial cellsfail to transform and to migrate, resulting in hypoplasticendocardial cushions [5]. Extensive remodeling and proliferationof the endocardial cushions occurs to form the adult heart valves.By E10.5, the right and left atria have divided [6]. The AVendocardial cushion region also plays an important role inseptation of the heart (Reviewed in [7]).Kru ¨ppel-like factor 2 (KLF2) is a member of a family of zincfinger-containing transcription factors [8–10]. In the E9.5 mouseheart, KLF2 mRNA is highly expressed in the endocardial cells ofthe AV cushion regions [11], where it is likely induced by highshear stress [12]. The role of KLF2 in cardiac development wasstudied by Lee et al., in knockout (KO) mice in a mixed geneticbackground including C57BL/6J. In KLF2 KO and KLF2endothelial conditional KO embryos, a cardiac phenotype isobserved as early as E10.5. These mice have thinning of themyocardium, high output heart failure, and die by E14.5. TheseKLF22/2 embryos also have cardiac functional defects due toloss of vessel tone, but no role for KLF2 in cushion formation wasnoted [11]. In two other KLF22/2 models, embryos in a mixedgenetic background die between E12.5 and E14.5, and exhibithemorrhaging [1,13]. The zebrafish gene, klf2a, a homolog ofKLF2 in mouse and man, is important in valve development.Knockdown of klf2a causes thicker and less flexible valves andPLOS ONE | www.plosone.org1 February 2013 | Volume 8 | Issue 2 | e54891

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increased regurgitation in the developing heart, compared to WT[14].In tissue culture, KLF2 plays a role as a molecular transducer offluid shear forces, thus directly or indirectly regulating a number ofendothelial genes [15,16]. Recent findings suggest that KLF2 playsan important role in endothelial barrier function in adult mice. Itpositively regulates expression of the tight junction proteinoccludin and modification of myosin light chain that is importantfor the integrity of the endothelial layer and to avoid vascularleakage [17]. In adult mice, KLF2 positively regulates theexpression of vasoprotective genes and inhibits expression ofpro-inflammatory genes in endothelial cells and macrophages,preventing atherosclerosis [18].klf2a knockdown results in abnormal zebrafish heart valvedevelopment, and KLF2 is expressed in the mouse endocardialcushion region. This suggests that KLF2 may be important inthe early stages of mammalian valve development. In this work,we show that FVB/N KLF22/2 hearts are hyperplastic withrespect to cells lining the AV canal, and hypoplastic withrespect to endocardial cushion mesenchymal cells. The datasuggests that KLF2 regulates EMT, and also atrial septation.KLF2 activates multiple important cardiovascular developmentgenes, suggesting mechanisms for its roles in the embryonicheart.Materials and MethodsEthics StatementThis study was approved by the Virginia CommonwealthUniversity Institutional Animal Care and Use Committee (VCUIACUC). VCU IACUC Protocol Number: AM10347.Generation of Knockout and Transgenic MiceThe traditional KLF2 KO mouse model was developed bytargeting the gene with the hypoxanthine phosphoribosyl-trans-ferase (Hprt) gene [13]. KLF2+/2 adults were mated with FVB/N or C57BL/6 mice for at least 12 generations to obtain KLF2+/2 animals in the FVB/N or C57BL/6 genetic background. Theseanimals were then mated to obtain KLF22/2 embryos. FVB/Nand C57BL/6 KLF2+/2 mice were mated to obtain KLF2+/2animals in a 50% FVB/N and 50% C57BL/6 genetic background(mixKLF2+/2). mixKLF2+/2 adults were mated to obtain mixWT and mix KLF22/2 embryos.Tie2-cre transgenic animals were purchased from JacksonResearch Laboratories (Bar Harbor, ME). Mice with a KLF2allele surrounded by loxP sites (floxed allele or KLF2fl/+)werekindly provided by Dr. Jerry Lingrel, University of Cincinnati andwere generated as previously described [19]. These animals weremated with FVB/N mice for at least 12 generations to obtainTie2-cre and KLF2fl/flanimals in an FVB/N genetic background.FVB/N Tie2-cre and KLF2fl/flanimals were then mated witheach other to obtain Tie2-cre, KLF2fl/+which were consequentlymated with KLF2fl/flanimals to obtain Tie2-cre, KLF2fl/flanimalsin the FVB/N background. These animals are designated FVB/NTie2-cre KLF22/2.Light and Electron Microscopic StudiesEmbryos were prepared for sectioning by fixing in 2%paraformaldehyde (PFA) and 0.25% glutaraldehyde, and embed-ded in eponate 12. Serial cross-sections of entire E9.5 and E10.5embryos of 7 and 5 mm, respectively, were obtained using an LKB2128 Ultramicrotome. Sections were stained with Toluidine Blue,and images were made using an Olympus BX41 compoundmicroscope and Olympus DP71 digital camera. E9.5 endocardialcell counts were obtained for the central section of the AV canalplus a section 2 sections anterior and a section 2 sections posterior.The ends of the AV canal were designated as 2 endothelial cellsbeyond the point where the AV canal widens into the ventricle orthe atria. E10.5 AV cushion mesenchymal cell counts wereobtained for the central section of all sections with endocardialcushion tissue associated with the AV canal. The counts wereexpressed in cells/mm2. Image J 1.46 software was used to tracethe irregular outline of the cushion and measure the area. Forelectron microscopy, embryos were sectioned at 100 nm with anLKB 2128 Ultratome and stained with 5% Uranyl Acetate andReynold’s Lead Citrate. Images were taken on a JEOL JEM-1230TEM with a Gatan Ultrascan 4000 digital camera.Benzidine StainingBenzidine staining was performed on cryosections of 4% PFA-fixed FVB/N WT and KLF22/2 embryos. Sections weresubmerged in 1X PBS for 1 hour prior to benzidine staining asdescribed previously [20]. Sections were then incubated inmethanol for 15 seconds, 1% benzidine in methanol for 5minutes, 2.5% hydrogen peroxide in 70% ethanol for 3 minutes,and washed with DI water for 2.5 minutes.ImmunohistochemistryFVB/N KLF2+/2 mice were mated to generate E10.5 WTand KLF22/2 embryos. Embryos were fixed in 4% PFA inMillonig’s buffer, washed in 1X Phosphate Buffered SalineTween-20 (PBST), and incubated in 0.5% hydrogen peroxide,0.5% normal goat serum (NGS) in PBST. The samples wereplaced in antibody blocking solution (10% goat serum in PBST)and incubated in primary antibody, PECAM (1:200) (BDBiosciences). The embryos were incubated with secondaryantibody, biotinylated Anti-Rat IgG (1:500) (Abcam) and BDPharmingen Streptavidin-Horseradish Peroxidase (Sav-HRP) andBD Pharmingen 3,39diaminobenzidine (DAB) chromogen inH2O2buffer and fixed in 4% PFA. The embryos were frozenand sectioned (10 mm) as described previously [21,22].Alcian Blue StainingE10.5 WT and KLF22/2 embryos were fixed in a 2% PFAand 0.25% glutaraldehyde solution and cryo-embedded. Cross-sections of 10 mm were cut using a vibratome (Ultrapro 5000).Sections were stained with Alcian Blue (Sigma Aldrich) andNuclear Fast Red (Sigma Aldrich) and observed using an OlympusBX41 microscope. Images were made using an Olympus DP71digital camera.qRT-PCR AssaysRNA was prepared from E10.5 AV canals, and quantitativeRT-PCR (qRT-PCR) was performed as previously described [22].Some of the primer sequences were as previously described byothers: hyaluronan synthase 2 or Has2 [23], Tbx5 [24] Notch1[25,26] and Gata4 [24]. The primer sequences for these and theother genes, PECAM1, Ugdh, Sox9 and Tgfb2, are given in TableS1. Mouse cyclophilin A mRNA was used as an internal standardfor normalization. mRNA amounts were quantified using SYBRGreen or Taqman reagents (Applied Biosystems). For assays usingSYBR Green chemistry, dissociation curves were generated, and itwas verified that only one product was amplified. A standard curvefrom pooled cDNA samples was included in each run to measurethe relative amounts of unknown samples. Statistical significancewas determined using the Student’s t-test.KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org2 February 2013 | Volume 8 | Issue 2 | e54891

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EchocardiographyKLF2+/2 adults were mated to obtain E10.5 embryos. Non-invasive in utero fetal ultrasound using a VisualSonics Vevo 770System and 40 MHz mechanical transducer (VisualSonics) wasperformed on 33 E10.5 embryos. Three pregnancies for the FVB/N and for the mixed genetic backgrounds were examined. Themice were anesthetized using pentobarbital (30 mg/kg; intraper-itoneal). The embryos were numbered for genotyping based ontheir position in utero, as described previously [11]. Maternaltemperature was maintained using a heat lamp, if required. The40 MHz VisualSonics RMV transducer gives an axial resolutionof 30 mm. For each embryo, blood flow parameters, includingheart rate, blood flow velocities and volumes were measured, aspreviously described [27]. Cardiac output was calculated as strokevolume multiplied by heart rate and expressed as beats per minute.2D imaging was used to view the 3 or 4 chamber heart andmeasure left ventricle ejection fraction. To statistically comparethe values in WT and KLF22/2, the Student’s t-test was used.AV Explant Collagen AssayAV regions were dissected from E10.5 FVB/N KLF22/2 andWT hearts and explants were placed on a collagen matrix aspreviously described [23,28–30]. After a 72 hour incubation, theexplants were observed and photographed using an OlympusIX70 inverted microscope with Hoffman Modulation Optics. Thecell counts were performed at a single plane of focus, at which thevast majority of the cells could simultaneously be observed.Chromatin Immunoprecipitation AssayChIP assays were performed essentially as described previously[31], using a KLF2 polyclonal antibody generated using apreviously described construct [32], and a negative controlpreimmune serum. Briefly, for each biological replicate, approx-imately 36106cells from ,8–10 E10.5 FVB/N WT AV regionswere cross-linked with 1% formaldehyde for 10 min at roomtemperature. Chromatin was sheared to approximately 500 bpusing a Bioruptor sonicator (Diagenode, Sparta, NJ). Chromatinwas precleared using protein G, precipitated with KLF2 orpreimmune antiserum, and cross-links were reversed. DNA waspurified and analyzed using quantitative PCR (qPCR) and SYBRGreen chemistry. Primer sequences for qPCR are indicated inTable S2. Fold enrichment was calculated as 2‘(Ctinput2 Cttest) andexpressed relative to the preimmune serum control.Statistical AnalysisThe Chi-square test, Student’s t-test or ANOVA were used forstatistical analyses, as indicated. Standard deviation was used tomeasure deviation from the mean, for all experiments. For all ofthe statistical tests, p values #0.05 were considered significant.ResultsKLF22/2 Embryos in the FVB/N Genetic Background Dieby E11.5Matings between FVB/N KLF2+/2 mice resulted in theexpected number of embryos of each genotype at E9.5 and E10.5.Out of 28 embryos from four KLF2+/2 matings, no (zero)KLF22/2 E11.5 embryos were obtained (expected frequen-cy=7). Chi-square analysis was performed to compare theobserved and expected frequencies (Table 1), and it wasdetermined that the number of KLF22/2 embryos wassignificantly less than expected (p=0.0044). FVB/N KLF22/2embryos die by E11.5, sooner than KLF22/2embryos in a mixedgenetic background, which die by E14.5 [1,11,13]. This suggeststhat modifier genes in FVB/N affect the KLF22/2 phenotype.E9.5 FVB/N KLF22/2 Mice have an Accumulation of CellsLining the AV CanalIn order to investigate the reasons for the earlier embryonicdeath of KLF22/2 mice in the FVB/N genetic background,serial sections of entire embryos were collected to assessmorphological abnormalities. Using light microscopy, anterior toposterior cross-sections of E9.5 FVB/N KLF22/2 embryos wereanalyzed. Compared to WT littermates, these KLF22/2embryos appear grossly normal, but at the cellular level there isa dramatic difference in the AV endocardial cushions. In WTembryos, the AV cushions are lined by a single layer of endothelialcells, as expected (Fig. 1A and 1C, n=3). However in the FVB/NKLF22/2 embryos, there is an increased number of cells liningthe AV canal region, and these cells form multiple disorganizedlayers (Fig. 1B and 1D, n=3). To determine whether the numberof endothelial cells in these E9.5 WT and KLF22/2 atrioven-tricular cushions is significantly different, cell counts wereperformed (Fig. 1E). There are 2-fold more cells lining the AVcanal in KLF22/2 than in WT. The Student’s t-test indicatesthat this is a significant difference with a p-value of 0.0078. AVmorphological defects were not reported in KLF22/2 heartsfrom mice in a mixed genetic background [1,11,13].FVB/N KLF22/2 Embryos have Abnormal EndocardialCell MorphologyThe AV cushion region defects in E9.5 FVB/N KLF22/2mice were studied at the subcellular level using transmissionelectron microscopy (TEM). TEM reveals that the E9.5 FVB/NWT (Fig. 2A) and the KLF22/2 (Fig. 2B) AV canals are patentand contain erythroid cells. The KLF22/2 AV canal is not linedby typical endocardial cells that are squamous (flat) but instead bycells that are bulbous with numerous cytoplasmic processesextending into the lumen of the AV canal (Fig. 2D). WT cellsare squamous and do not have projections or have projectionstowards the cushions, rather than lumen, as expected (Fig. 2C).Table 1. Number of embryos observed and expected from FVB/N KLF2+/2 matings.StageTotal viableembryos WT OWT E KLF2+ +/2 2 O KLF2+ +/2 2 EKLF22 2/2 2 O KLF22 2/2 2 E Chi-square p-valueE9.5 268 6.5 12 136 6.50.7939E10.52987.25 1414.577.250.9496E11.52712 (0)6.7515 (1)13.50 (2)6.75 0.0044*Parentheses indicate number of dead embryos at E11.5. O is observed; E is expected. Asterisk indicates a statistically significant difference between O and E.doi:10.1371/journal.pone.0054891.t001KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org3 February 2013 | Volume 8 | Issue 2 | e54891

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Moreover, additional cells lie adjacent to the endocardial layer inKLF22/2 compared to WT, which makes the cushions appeardisorganized and non-laminar.E10.5 FVB/N KLF22/2 Embryos have Hypoplastic AVEndocardial Cushions and Other Cardiac AbnormalitiesTo examine the KLF22/2 AV endocardial phenotype at alater stage of development, light microscopy was performed atE10.5 (n=3). The cardiac abnormalities in FVB/N KLF22/2are more severe at E10.5 than at E9.5. The cells in the AV canalregion and endocardial cushions in FVB/N KLF22/2 (Fig. 3Cand 3D) are disorganized compared to somite matched FVB/NWT controls (Fig. 3A and 3B). In E10.5 FVB/N KLF22/2embryos, the AV cushions are hypocellular (Fig. 3D at *)compared to FVB/N WT which are highly populated withmesenchymal cells (Fig. 3B at *). In FVB/N KLF22/2, theendocardial cells are evidently unable to transform into mesen-chymal cells and migrate into the cushions, and thereforeendothelial-like squamous cells accumulate lining the AV canal.Moreover, the E10.5 FVB/N KLF22/2 heart has only oneatrium (Fig. 3C), whereas somite-matched FVB/N WT heartshave a left and right atrium at this time point (Fig. 3A), indicatingthat there is also an atrial septal abnormality in the septumprimum in the mutants. Additionally, in the E10.5 FVB/NKLF22/2 heart (Fig. 3C), the myocardium is thinner than in theFVB/N WT heart (Fig. 3A), as previously reported [11]. KLF2 isnot expressed in the myocardium, thus its effect on myocardialdevelopment must be indirect.E10.5 endothelial-specificTie2-creKLF22/2) hearts show hypocellular endocardial cushions,KLF22/2(Tie2-credisorganized AV canal regions, and delayed atrial septal formation(Fig. 3G and 3H), similar to the traditional KLF2 KO. Using thismodel, the deletion of the KLF2 gene in the heart is quitecomplete at approximately 84% (D. Vinjamur, unpublished data).Negative control littermates having the floxed KLF2 gene withoutTie2-cre are unaffected, as expected and shown in Fig. 3E and 3F.This suggests that KLF2 has an endothelial cell-autonomous rolein the AV cushion region.The E10.5 FVB/N KLF22/2 heart morphological phenotypeis more severe than that previously reported by Lee et al. for micein a mixed genetic background, which had only myocardialthinning [11]. To confirm that the KLF22/2 phenotype varies indifferent genetic backgrounds, matings were carried out to obtainKLF22/2 embryos in a controlled genetic background that is50% FVB/N and 50% C57BL/6 (mix KLF22/2). Lightmicroscopy studies on E10.5 mix KLF22/2 indicate that theAV canal and cushion morphology in these embryos (Fig. 3K and3L) is comparable to somite- and genetic background-matchedmix WT embryos (Fig. 3I and 3J). The endocardial cushions inmix KLF22/2 embryos do not have a drastic reduction inmesenchymal cells (Fig. 3L). However, mix KLF22/2 embryos(Fig. 3K) have thinner myocardium than mix WT (Fig. 3I), aspreviously reported [11]. C57BL/6 KLF22/2 hearts (Fig. 3Oand 3P) have apparently normal AV cushions like mix KLF22/2and C57BL/6 WT (Fig. 3M and 3N), and thinner myocardiumlike mix KLF22/2 and FVB/N KLF22/2. C57BL/6 KLF22/2 hearts (Fig. 3O) have atrial septal abnormality similar to thatobserved in FVB/N KLF22/2 (Fig. 3C), but not found in mixKLF22/2 (Fig. 3K). To quantify the mesenchymal cellhypocellularity of the AV endocardial cushions, cell counts wereFigure 1. E9.5 FVB/N KLF22 2/2 2 atrioventricular cushions have accumulated cells lining the AV canal. (A) and (C) are WT (n=4) and (B)and (D) are KLF22/2 (n=4) hearts. Micrographs A and B (magnification 50X) show atrial (At) and ventricular (V) chambers. The boxes or bracketsenclose the endocardial cushions (EC) shown at higher magnification in C and D (400X). Endo: Endocardial cells; Myo: Myocardium; Ery: erythroid cells;Mes: Mesenchymal cells. (E) Bar chart representing the number of cells/mm2 lining the AV canal of E9.5 WT and KLF22/2 embryos. The Student’s t-test indicates that the number of cells is significantly greater in KLF22/2 than in WT (p=0.0078). n=4.doi:10.1371/journal.pone.0054891.g001KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org4 February 2013 | Volume 8 | Issue 2 | e54891

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performed for E10.5 WT and KLF22/2 embryos in the FVB/N,C57BL/6 and mixed genetic backgrounds, and for the FVB/NTie2-cre KLF22/2 (Fig. 3Q). As expected, FVB/N KLF22/2and Tie2-cre KLF22/2 showed highly significant (p,0.001)decreases in the number of mesenchymal cells in the endocardialcushion tissue associated with the AV canal, compared to controls.Interestingly, C57BL/6 KLF22/2 embryos also showed astatistically significant (p=0.03) decrease in the mesenchymal cellcount, suggesting that there is some role for KLF2 in EMT in thisstrain. There may be unique recessive modifier genes involved inthe decreased AV mesenchymal cell numbers in FVB/N and inC57BL/6 KLF22/2 hearts, because the number is normal inmix KLF22/2 hearts.Unlike WT (Fig. 4A), in FVB/N KLF22/2 embryos, erythroidcells are observed outside of the dorsal aortas, suggesting possiblehemorrhaging (Fig. 4B and 4C). Alternatively, the presence oferythroid cells outside of the FVB/N KLF22/2 dorsal aortasmay be due to an inability of hematopoietic progenitors,originating in the para-aortic mesenchyme, to reach the aorticendothelium and enter circulation [33]. To confirm that these cellsare erythroid, benzidine staining was performed. In FVB/NKLF22/2 embryos, there are benzidine-positive cells in the tissuesurrounding the dorsal aorta (Fig. 4E), but they are found onlywithin the dorsal aorta in FVB/N WT (Fig.4D). These results werereplicated in E10.5 FVB/N WT and KLF22/2 hearts at the 34and 36 somite stages. There are no blood cells in the tissuessurrounding the dorsal aortas in mix KLF22/2 embryos (Fig. 4Gand 4H), which look comparable to mix WT (Fig. 4F) and FVB/NWT (Fig. 4A).The outflow tract endocardial cushions in two out of threeE10.5 FVB/N KLF22/2 embryos examined had an abnormal,non-laminar accumulation of endocardial cells lining the lumen.These same embryos also had a smaller number of mesenchymalcells in the outflow tract cushions than WT (Fig. S1). Therefore,although the outflow tract phenotype is not completely penetrant,there are similarities between the AV and outflow tract phenotypesin FVB/N KLF22/2 mice.Cells Accumulated in the FVB/N KLF22/2 AV Canal haveEndothelial CharacterTo further define the role of KLF2 in EMT, the cellsaccumulating in the FVB/N KLF22/2 AV canal were studied.Immunohistochemical staining was performed using the endo-thelial specific PECAM (CD31) antibody. The cells accumulatedin the FVB/N KLF22/2 AV canal are CD31 positive,indicating that they have endothelial characteristics (results fromtwo embryos shown in Fig. 5C–5F). The FVB/N WT positivecontrol has an organized layer of CD31 positive endothelialcells lining the AV canal (Fig. 5A and 5B). A negative control,Figure 2. Electron microscopy of E9.5 FVB/N KLF22 2/2 2 atrioventricular cushions shows abnormal endocardial cell morphology. InWT (A), the endocardium is one cell-layer thick but in KLF22/2 (B), there are multiple disorganized cell layers. The boxes enclose the AV canal. Athigher magnification, endocardial cells in KLF2 KO (D) extend cytoplasmic projections or filopodia-like extensions towards the lumen of the AV canal,which are not observed in WT (C). Arrowhead indicates abnormal cytoplasmic projection towards lumen in KLF22/2. *indicates non-laminar cellsaccumulated at the lining of the AV canal that are observed in KLF22/2 but not in WT.doi:10.1371/journal.pone.0054891.g002KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org5 February 2013 | Volume 8 | Issue 2 | e54891

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Figure 3. E10.5 FVB/N but not mix and C57BL/6 KLF22 2/2 2 atrioventricular endocardial cushions are hypoplastic and disorganized.(A) and (B) are micrographs of FVB/N WT heart; (C) and (D) are micrographs of FVB/N KLF22/2 heart. The light micrographs, A and C (magnification100X), show the structure of the E10.5 heart including the atrial (At) and the ventricular chambers (V) and B and D show the endocardial cushionregions that are in the boxes in A and C respectively, magnified at 200X. The red dashed lines indicate the positions of the hypoplastic FVB/N KLF22/KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org6 February 2013 | Volume 8 | Issue 2 | e54891

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reactedExpression of PECAM1 mRNA was significantly higher inFVB/N KLF22/2 than in FVB/N WT AV canals as shownby quantitative reverse transcriptase-PCR (qRT-PCR, Fig. 5G).This suggests that there is an increased number of cellsexpressing PECAM1mRNA, and/or PECAM1 expression percell is increased in KLF22/2. However, because an accumu-onlywith secondary antibody,had nostaining.lation of endothelial-like squamous cells lining the AV canal isobserved in KLF22/2, there is a high probability that thereare a higher number of cells expressing PECAM1 mRNA.These findings support the premise that there is abnormal EMTin the AV cushions of FVB/N KLF22/2 embryos and thatthe cells abnormally accumulating at the AV canal areendothelial cells that are unable to transform.2 AV cushions in D, compared to normal AV cushions in H and L. (G) and (H) are micrographs of Tie2-cre KLF22/2, and (E) and (F) are WT littermatecontrols without Tie2-cre; all are in the FVB/N background. (I) and (J) are micrographs of mix WT; (K) and (L) are micrographs of mix KLF22/2 hearts.(M) and (N) are micrographs of C57BL/6 WT; (O) and (P) are micrographs of C57BL/6 KLF22/2 hearts. (Q) Bar chart representing the number ofmesenchymal cells/mm2in the endocardial cushion tissue associated with the AV canal. Counts were performed of all cells in a single central sectionfrom E10.5 WT or KLF22/2 hearts in all three genetic backgrounds and for Tie2-cre KLF22/2. Student’s t-test indicates that the number ofmesenchymal cells is decreased in FVB/N KLF22/2, Tie2-cre KLF22/2 and C57BL/6 KLF22/2, compared to WT. Mes: Mesenchymal cells; Endo:Endothelial cells; Ery: Erythroid cells; Myo: Myocardium. n=3–5 hearts for histological staining, and n=3 hearts for mesenchymal cell counts. WT andKO embryos for each background are somite matched. Asterisks* indicate AV endocardial cushion region. Arrowheads show atrial septum that isforming in A, E, I, K and M; the atrial septum is absent in C, G and O.doi:10.1371/journal.pone.0054891.g003Figure 4. E10.5 FVB/N KLF22 2/2 2 but not mix KLF22 2/2 2 embryos have erythroid cells outside of the dorsal aortas. (A) is a lightmicrograph (100X) of the FVB/N WT E10.5 dorsal aortas (DA). Erythroid cells (Ery) are found only within the aortas. (B) FVB/N KLF22/2 have a numberof erythroid cells in tissue surrounding the DA. The box indicates the region that is magnified in C (200X). (C) Erythroid cells within and outside thevessel in FVB/N KLF22/2. (D) and (E) are light micrographs of benzidine-stained FVB/N WT and KLF22/2 sections, respectively (400X). Ery indicatesbenzidine-positive, brown-colored erythroid cells. Mes indicates mesenchymal cells. (F), (G) and (H) are mix WT (100X), mix KLF22/2 (100X) and mixKLF22/2 (200X), respectively, showing normal aortas with erythroid cells within the vasculature only. (n=3 each).doi:10.1371/journal.pone.0054891.g004KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org7 February 2013 | Volume 8 | Issue 2 | e54891

encoding enzymes involved in the synthesis of glycosaminoglycans,quantitative RT-PCR was performed using RNA from WT andKLF22/2 AV canals. As shown in Fig. 6E, the expression ofUDP-glucose dehydrogenase (Ugdh) mRNA was significantlyreduced in FVB/N KLF22/2 compared to FVB/N WT, butsimilar amounts were expressed in mix WT and mix KLF22/2.This correlates with the reduced alcian blue staining in FVB/NKLF22/2, but not in mix KLF22/2, compared to WT. UGDHconverts UDP-glucose to UDP-glucuronic acid, which is requiredfor the biosynthesis of GAG components like hyaluronan, heparinsulfate and chondroitin [34]. Expression of the hyaluronansynthase 2 (Has2) gene was not reduced in FVB/N KLF22/2compared to WT AV canals (data not shown). Therefore areduction in Has2 is not likely to be the cause of the reducedGAGs observed in FVB/N KLF22/2 hearts.A Reduced Number of Mesenchymal Cells Invade theCollagen Gel in FVB/N KLF22/2 AV Explant AssaysTo determine whether the EMT defect is caused solely by adefect in the cardiac jelly, or also by an abnormality in theendocardial cells in FVB/N KLF22/2 hearts, E10.5 AV explantassays were performed using a collagen gel. FVB/N WT explantsunderwent EMT and the mesenchymal cells migrated into thecollagen matrix during the 72 hour incubation (Fig. 6F). Com-pared to WT explants, KLF22/2 explants showed a significantreduction in the number of mesenchymal cells that migrated intothe collagen matrix (Fig. 6G and 6H). The KLF22/2 explantshave at least a 10-fold reduction in the number of transformedcells compared to WT (Fig. 6H). In addition, the fraction ofmigrated cells that are transformed is greater than activated cells inWT explants, but the reverse is true in KLF22/2. This suggeststhat a defect in the endocardial cells, as well as in the cardiac jellycomposition, is responsible for abnormal EMT in the FVB/NKLF22/2 AV cushions.FVB/N KLF22/2 Hearts Exhibit Abnormal CardiacFunctionEchocardiography was performed on E10.5 FVB/N WT, FVB/N KLF22/2, mix WT and mix KLF22/2 embryos. The heartrate was not significantly different in embryos of the 4 genotypes(Fig. S3A), but cardiac output and ejection fraction are signifi-cantly higher in FVB/N KLF22/2 than in WT embryos (Fig.S3B and S3C left side, respectively). The descending aorta velocityin FVB/N KLF22/2 hearts is significantly lower than WT (Fig.S3D left side). Higher than normal cardiac output and ejectionfraction were observed in E11.5 KLF22/2 embryos in a mixedgenetic background [11], but these features were not evident inE10.5 mix KLF22/2 (Figs. S3B, S3C and S3D; right side). It isdifficult to reconcile the specific abnormalities in the heartparameters in FVB/N KLF22/2 with the observed morpholog-ical defects.Cardiovascular Genes Important for AV CushionDevelopment and Septation are Downregulated in FVB/N KLF22/2 Compared to WT AV RegionsKLF2 is a transcription factor expressed in endothelial cells. Tobegin to elucidate the molecular mechanism by which KLF2ablation results in heart development and septation abnormalities,the amounts of expression of candidate genes important in AVcushion development (Tbx5, Sox9, Tgfb2, Notch1, Gata4) andatrial septation (Tbx5, Gata4) (Reviewed in [7]) were quantified inAV regions dissected from FVB/N WT and KLF22/2 hearts.Each of these genes has at least one consensus KLF2 binding sitein its promoter (CCACCC and CCGCCC) [10,32], within 500 bpupstream of the transcription start site (Table S3).The mRNAs for three cardiovascular transcription factors,Tbx5, Gata4 and Sox 9, showed significantly reduced expressionin FVB/N KLF22/2 compared to WT AV regions (Figs. 7A–7C), indicating that these genes are, directly or indirectly,positively regulated by KLF2. FVB/N Tie2-cre KLF22/2 AVcanals show a similar and significant reduction in the expression ofthe Tbx5, Gata4 and Sox9 genes, compared to controls withoutTie2-cre (Fig. S4). Interestingly, expression of these genes is notreduced in mix KLF22/2 compared to mix WT, consistent withthe absence of the AV cushion and atrial septal phenotypes in mixKLF22/2. Apparently, strain-specific modifier genes differen-tially affect the expression of Ugdh, Sox9, Tbx5 and Gata4 mRNAin response to KLF2 ablation. Other roles of Gata4 and Tbx5 arediscussed in detail later, but Gata4 and Tbx5 double heterozygousknockout mice show myocardial thinning [24,35,36], likeKLF22/2. Mix KLF22/2 has myocardial thinning but noreduction of Gata4 and Tbx5 mRNA, indicating that anadditional unknown gene(s) is related to this phenotype. Sox9plays an important role during endocardial cushion EMT as wellas valve remodeling [37]. The Notch1 and Tgfb2 genes areimportant for atrioventricular development, but these mRNAs arenot expressed significantly differently in KLF22/2 and WThearts, whether the animals are in an FVB/N or a mixed geneticbackground (data not shown). The data suggest that Tbx5, Gata4,Sox9 and Ugdh are downstream of KLF2 and have a geneticbackground specific role in AV endocardial cushion EMT, atrialseptation and cardiac jelly synthesis. More than one cardiovasculargene shows reduced expression by 2–3 fold in the absence ofKLF2, suggesting that the KO phenotype is an outcome ofdysregulation of multiple downstream targets of KLF2.KLF2 Binds to the Mouse Gata4, Tbx5 and UgdhPromotersKLF2 positively regulates mRNA expression of Gata4, Tbx5,Sox9 and Ugdh. To better understand the mechanism of KLF2regulation, chromatin immunoprecipitation (ChIP) assays using aKLF2 polyclonal antibody [31] were performed using cells fromE10.5 FVB/N WT mouse AV regions. Due to the limitedavailability of tissue per sample, between eight and ten E10.5 AVregions were pooled for each ChIP assay. The Gata4, Tbx5 andSox9 promoters have multiple potential KLF2 binding sites, andthe Ugdh promoter has a single site (Table S3). Due to assaylimitations, the two Tbx5 and two Sox9 binding sites could not bedistinguished from each other, and were tested simultaneously.Two regions of the Gata4 promoter, each containing two putativeKLF2 binding sites (designated 2100 and 2411), were tested forKLF2 enrichment. Quantitative PCR (qPCR) was used todetermine the fold-enrichment of KLF2 at each promoter region,by comparing ChIP assays with KLF2-antiserum to pre-immuneserum. The data in Figure 7D indicate that KLF2 showed anapproximately 70-fold enrichment at the Tbx5 promoter, 25-foldenrichment at the Ugdh promoter, and 15-fold enrichment at theproximal Gata4 promoter site (2100) compared to negativecontrol assays using pre-immune serum. No significant KLF2enrichment was observed at the Sox9 promoter. As a negativecontrol, KLF2 does not bind to the promoter of the b-actin gene.The ChIP assays thus indicate that KLF2 binds to the Gata4,Tbx5 and Ugdh promoters, and therefore may directly regulatethese genes. No evidence was obtained to indicate that KLF2directly regulates the Sox9 gene.KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org9February 2013 | Volume 8 | Issue 2 | e54891

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DiscussionCardiovascular development and morphogenesis is a complexprocess, involving a number of highly conserved transcriptionfactors and signaling pathways [38]. KLF2 plays a multi-facetedrole in cardiovascular development. It is expressed in theendocardium of the developing heart. The accumulation ofendothelial-like cells lining the AV canal, reduced EMT, delayedatrial septal formation, and the absence of normal cardiac jellycomposition are novel phenotypes for FVB/N KLF22/2 mice,and therefore may be related to the earlier embryonic death in theFVB/N genetic background. Our studies also identify putativeeffectors downstream of KLF2 that may impact each of theseprocesses in the embryonic heart.KLF2 is important for vascular integrity [17]. Kuo et al. [1] andWani et al. [13] reported hemorrhaging in the abdominal andcardiac outflow tract region in KLF22/2 embryos. To thecontrary, no hemorrhaging was observed in the KLF22/2embryos examined by Lee et al. [11]. This discrepancy may bepartially explained by the current study. Erythroid cells wereobserved outside E10.5 KLF22/2 dorsal aortas in FVB/N butnot in mixed genetic background embryos, indicating that thisphenotype is genetic background-specific. The genetic backgroundof the KLF22/2 mice used in the previous studies was not welldefined. It is theoretically possible that the dorsal aorta defect inFigure 6. E10.5 FVB/N KLF22 2/2 2 hearts have abnormal cardiac jelly, and a reduction in transformed mesenchymal cells. Alcian bluestaining for extracellular matrix and counterstain with nuclear fast red was performed on cross-sections of E10.5 FVB/N WT and KLF22/2 hearts(n=3). (A) and (C) are WT AV cushions; the nuclei are stained red and the extracellular matrix is stained blue (100X and 200X magnifications,respectively). (B) and (D) are FVB/N KLF22/2 AV cushions with decreased blue staining, indicating reduced glycosaminoglycans (100X and 200Xmagnifications, respectively). At: Atrium; V: Ventricle. Boxes indicate the AV endocardial cushion region. (E) qRT-PCR indicates a 2-fold decrease inexpression of Ugdh mRNA (p=0.0204) in FVB/N KLF22/2 but not mix KLF22/2 compared to WT AV region (n=7). (F) and (G) AV canal explantswere incubated in vitro on a collagen matrix for 72 hours, and the cells migrating into the matrix were observed (100X magnification). (F) FVB/N WTexplants show mesenchymal cells migrating into the collagen matrix (n=5). Arrows indicate mesenchymal cells. Round cells are activated but nottransformed. Stellate cells are activated and transformed into mesenchymal cells. (G) FVB/N KLF22/2 explants have less mesenchymal cellsmigrating into the collagen matrix than WT (n=5), indicating an EMT defect in the FVB/N KLF22/2 endocardial cells. (H) The bar chart indicatespercentage of transformed cells in FVB/N WT and FVB/N KLF22/2 (n=3). The number of cells in WT is designated as 100%. KLF22/2 explants have agreater than 10-fold decrease in mesenchymal cells in the collagen matrix compared to WT (p,0.001).doi:10.1371/journal.pone.0054891.g006KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org10 February 2013 | Volume 8 | Issue 2 | e54891

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FVB/N KLF22/2 embryos causes shear stress, resulting in theAV cushion defect. However, this hypothesis is not favoredbecause the AV cushion defect is observed by E9.5, whereas thedorsal aorta defect is first apparent at E10.5.The importance of genetic background in cardiac developmenthas been demonstrated in a number of studies. Sakata et al.studied Hey2 deficient mice and observed a spectrum ofcardiovascular anomalies that varied in the BALB/c andC57BL/6 genetic backgrounds [39]. Astrof et al. studied the roleof fibronectin in heart development; a null mutation in the generesults in arrested heart development earlier in 129S4 than inC57BL/6 embryos [40]. The current study shows that the role ofKLF2 in the morphology and function of the developing heart isalso genetic background specific. In the FVB/N background, lossof KLF2 results in an EMT defect in the AV cushion region,delayed formation of the atrial septum, myocardial thinning anddeath by E10.5. In the C57BL/6 background, KLF22/2 showsdelayed atrial septation and myocardial thinning. In a mixedbackground the major defect in KLF22/2 hearts is myocardialthinning [11].In this work, we have demonstrated that KLF2 binds thepromoters of, and positively regulates, the Tbx5 and Gata4 genesin the mouse E10.5 AV region. An endocardial specific Gata4 KOhas multiple layers of endocardium in the AV canal, andhypocellular AV cushions at E10.5 [41], similar to FVB/NKLF22/2. Tbx5 KO embryos have hypoplastic endocardialcushions [42], like FVB/N KLF22/2. These phenotypescorrelate with our observation that Gata4 and Tbx5 expressionis reduced 3-fold in the absence of KLF2 in FVB/N AV canals.Interestingly, the Tbx5 and Gata4 proteins physically interactduring cardiac development. A heterozygous mutation (mG295S)in the Gata4 gene disrupts these protein interactions, resulting incardiac defects like atrial septal defects (ASD), AV septal defects(AVSD) and myocardial thinning beginning at E11.5 [35,36]. TheAVSD and ASD in these mice are known to result from abnormalEMT and remodeling of endocardial cushions. Gata4 mG295S isFigure 7. Cardiovascular genes are dysregulated in FVB/N KLF22 2/2 2 AV region. Quantitative RT-PCR (qRT-PCR) of AV region RNA was usedto test the amount of expression of genes important for AV cushion development (Tbx5 and Sox9) and atrial septation (Tbx5 and Gata4). CyclophilinA mRNA was used as a normalization control. FVB/N WT and mix WT were designated as100%, and KLF22/2 were compared to WT. (A–C) In E10.5FVB/N KLF22/2 AV region there is significantly decreased expression of (A) Tbx5 (p=0.0175), (B) Gata4 (p=0.0164) and (C) Sox9 (p=0.019) mRNAcompared to FVB/N WT, but Mix WT and Mix KLF22/2 hearts have no differences in expression of these genes. Error bars indicate standard deviation.n=5. D) ChIP assays were performed on cells obtained from E10.5 WT AV regions. Approximately 8 to 10 WT AV regions were pooled to obtain cellsfor one replicate. Polyclonal anti-KLF2 and non-specific control pre-immune serum was used. The y-axis represents the relative fold-enrichment. Themean pre-immune enrichment was designated as ‘1.0’ and the enrichment with KLF2 antisera was scaled appropriately. The x-axis indicates thelocation of the primers used for qPCR; all were in gene promoters and are described in Table S2. Pr: Promoter. Primers specific for the b-actin genewere used as a negative control. n=7 biological replicates.doi:10.1371/journal.pone.0054891.g007KLF2 Is Required in Mouse Cardiac DevelopmentPLOS ONE | www.plosone.org 11February 2013 | Volume 8 | Issue 2 | e54891

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"This type of dual analysis of embryo hearts has been conducted in zebrafish (e.g., Vermot et al., 2009) and has promise in avian systems (e.g., Groenendijk et al., 2004, 2007; Bressan et al., 2013). For the mouse model, echocardiography of embryos is the technique most often paired with the molecular studies (e.g., Chiplunkar et al., 2013; Rog-Zielinska et al., 2013). The ideal situation would be to have a marker of molecular changes that can be accessed without affecting physiological function. "

[Show abstract][Hide abstract]ABSTRACT: Disturbed cardiac function at an early stage of development has been shown to correlate with cellular/molecular, structural as well as functional cardiac anomalies at later stages culminating in the congenital heart defects (CHDs) that present at birth. While our knowledge of cellular and molecular steps in cardiac development is growing rapidly, our understanding of the role of cardiovascular function in the embryo is still in an early phase. One reason for the scanty information in this area is that the tools to study early cardiac function are limited. Recently developed and adapted biophotonic tools may overcome some of the challenges of studying the tiny fragile beating heart. In this chapter, we describe and discuss our experience in developing and implementing biophotonic tools to study the role of function in heart development with emphasis on optical coherence tomography (OCT). OCT can be used for detailed structural and functional studies of the tubular and looping embryo heart under physiological conditions. The same heart can be rapidly and quantitatively phenotyped at early and again at later stages using OCT. When combined with other tools such as optical mapping (OM) and optical pacing (OP), OCT has the potential to reveal in spatial and temporal detail the biophysical changes that can impact mechanotransduction pathways. This information may provide better explanations for the etiology of the CHDs when interwoven with our understanding of morphogenesis and the molecular pathways that have been described to be involved. Future directions for advances in the creation and use of biophotonic tools are discussed.

[Show abstract][Hide abstract]ABSTRACT: The Kruppel-like factor (KLF) family of transcription factors regulates diverse biological processes that include proliferation, differentiation, apoptosis, development, and responses to external stress. In the present study, we aim to investigate the roles of KLF2 in the hepatic steatosis. Our resuls showed that mRNA and protein levels of KLF2 were significantly elevated in livers from obese mice. Adenoviruses-mediated overexpression of KLF2 induced accumulation of triglycerides in C57BL/6 mice, while KLF2 silencing ameliorates hepatosteatosis in ob/ob mice. At the molecular level, our data established CD36 as a novel transcriptional target of KLF2. KLF2 upregulated CD36 expression through a consensus binding site on its proximal promoter region. Additionally, the steatotic effect of KLF2 was dramatically inhibited in CD36 null mice. Therefore, our study reveals a novel link between KLF2-induced hepatic triglycerides accumulation and the expression of CD36.

[Show abstract][Hide abstract]ABSTRACT: In mammalian ES cells, the transcription factors Klf4 and Klf2 contribute to maintenance of pluripotency and self-renewal and are regulated by Pou5f1/Oct4. In the early zebrafish embryo Pou5f1/Oct4 is necessary for expression of three Klf2/4 family members, klf2a, klf2b and klf17 (previously klf4b), similar to the regulation reported for mammalian ES cells. In this study, we analyzed blastula and gastrula stage Klf regulatory networks and their influence on zebrafish embryonic patterning. We show that Pou5f1 acts in combination with region-specific factors to activate klf2a, klf2b, and klf17 in the superficial cell layer of the embryo. In addition, Pou5f1 acts together with the BMP signaling pathway to activate and maintain expression of klf2a and klf2b in a ventral ectodermal domain. We used microarray expression profiles of klf2a, klf2b and klf17 knockdown and overexpression embryos to identify Klf target genes, which reveals that Klfs participate in specification of the extraembryonic enveloping layer (EVL). We discuss mechanistic implications of simultaneous activation of transcriptional targets by ubiquitous, like Pou5f1, and region-specific inducers, emerging as a common regulatory motif in early development.